Cancer remains a major health burden worldwide. The aggressive targeting of metabolic pathways shared by normal and cancer cells results in prolonged survival and cures, but at a tremendous cost to patient life quality. What is missing is a therapeutic agent that clearly differentiates normal and cancer cells. This proposal delineates a process for killing exclusively cancer cells with no interference with normal cells. In the past two decades there has been considerable effort to develop attenuated viruses for killing cancer cells. Of the oncolytic viruses in clinical trials, attenuated herpes simplex viruses (HSV) are among the most promising because of safety, affinity for cancer cells, ability to treat patients multiple times without block by adaptive immunity. The shortcoming is that they do not discriminate between normal and cancer cells, are effective in a limited number of patients. The remarkable accomplishment by my laboratory at the basis of the proposal is the genetic engineering of HSVs that specifically infect and kill cancer cells and cannot infect normal cells. The prototype retargeted HSV targets HER2, a receptor in breast, ovary and other tumors. HER2-HSV ablates human breast and ovary cancers, and glioblastoma after intratumoral or intraperitoneal administration.This proposal addresses basic research issues for the advancement of retargeted oncolytic HSVs. It is organized in 5 AIMS• Engineer a non-cancer cell line for virus production acceptable to Health Authorities and re-engineer the retargeted-HSV accordingly. This will enable production of clinical grade retargeted-HSVs for clinical tests (AIM1)• Engineer retargeted-HSVs suitable for systemic delivery and for boosting anti-tumor immunity (AIM2-3)• Apply our platform to expand the repertoire of oncolytic HSVs that target glioblastomas, prostate, head-and-neck, colon carcinomas (AIM4)• Determine the tumorigenic potential of cancer cells that escape killing by retargeted HSVs (AIM5)